The rapid development of CRISPR-Cas gene editing technologies has revolutionized genetic medicine, offering unprecedented precision and potential for treating a wide array of genetic disorders. However, assessing the risks of unintended gene editing effects remains critical, and is complicated by new editing modalities and unclear analytical guidelines. We present UNCOVERseq (Unbiased Nomination of CRISPR Off-target Variants using Enhanced RhPCR), an improved in cellulo off-target nomination workflow designed to sensitively nominate off-target sites (<0.01% editing) with defined input requirements and analytical process controls to provide empirical performance evidence across diverse circumstances. Using this workflow, we nominated off-targets across 192 guide RNAs (gRNAs) and demonstrated superior performance compared to existing methodologies. We identified a subset of six gRNAs with a dynamic range of specificity and confirmed the relevance and high true positive rate of our nomination method, providing relative risk assessments for multiple modalities (S.p. Cas9 and derived high-fidelity variants / base editors) in a translational system involving hematopoietic stem and progenitor cells (HSPCs). Additionally, we established that double-strand break (DSB) editing retains a strong, positive rank correlation to single-strand break (SSB)-mediated base editing, highlighting the importance of DSB nomination sites as candidate loci for base editing. Overall, UNCOVERseq improves informed risk assessment of gene editing in translational systems by enhancing the quality of off-target nomination.

We demonstrate here the use of optical genome mapping (OGM) to detect genetic alterations arising from gene editing by various technologies in human induced pluripotent stem cells (iPSCs). OGM enables an unbiased and comprehensive analysis of the entire genome, allowing the detection of genomic structural variants (SVs) of all classes with a quantitative variant allele frequency (VAF) sensitivity of 5%. In this pilot study, we conducted a comparative dual analysis between the parental iPSCs and the derived cells that had undergone gene editing using various techniques, including transposons, lentiviral transduction, and CRISPR-Cas9-mediated safe harbor locus insertion at the adeno-associated virus integration site 1 (AAVS1). These analyses demonstrated that iPSCs that had been edited using transposons or lentiviral transduction resulted in a high number of transgene insertions in the genome. In contrast, CRISPR-Cas9 technology resulted in a more precise and limited transgene insertion, with only a single target sequence observed at the intended locus. These studies demonstrate the value of OGM to detect genetic alterations in engineered cell products and suggests that OGM, together with DNA sequencing, could be a valuable tool when evaluating genetically modified iPSCs for research and therapeutic purposes.

Gene editing technologies have opened the possibility of directly targeting viral DNA in therapeutic applications. In chronically infected hepatocytes with hepatitis B virus (HBV), covalently closed circular DNA (cccDNA) serves as the master template for viral transcripts and gene products. In the present study, we evaluated the outcomes of anti-HBV multiplex gene editing with the CRISPR-Cas9 endonuclease from Staphylococcus aureus (SaCas9) using primary human hepatocytes (PHHs) and HBV mouse models. Nonviral delivery of SaCas9-encoding mRNA and a pair of HBV-targeting guide RNAs (gRNAs) substantially reduced viral biomarkers and intrahepatic HBV DNA copies in vitro and in vivo, suggesting that fragmentation of HBV DNA primarily leads to its degradation. Hybridization capture sequencing analyses indicated that small insertions and deletions (indels) and structural variants including excisions and inversions of the viral sequences were accumulated in the residual HBV DNA. These assays also demonstrated that transient expression of the HBV-targeting SaCas9 significantly suppressed random integration of HBV DNA, while this therapeutic approach was unlikely to affect chromosomal translocations involving viral copies. Taken together, our results suggest that anti-HBV multiplex gene editing eliminates viral DNA from chronically infected hepatocytes, potentially reducing the risk of hepatocarcinogenesis associated with HBV DNA integration.

CRISPR-Cas is a defense system of bacteria and archaea against phages. Parts of the foreign DNA, called spacers, are incorporated into the CRISPR array which constitutes the immune memory. The orientation of CRISPR arrays is crucial for analyzing and understanding the functionality of CRISPR systems and their targets. Several methods have been developed to identify the orientation of a CRISPR array. To predict the orientation, different methods use different features such as the repeat sequences between the spacers, the location of the leader sequence, the Cas genes, or PAMs. However, those features are often not sufficient to predict the orientation with certainty, or different methods disagree. Remarkably, almost all CRISPR systems have been found to insert spacers in a polarized manner at the leader end of the array. We introduce CRISPR-evOr , a method that leverages the resulting patterns to predict the acquisition orientation for (a group of) CRISPR arrays by reconstructing and comparing the likelihood of their evolutionary history with respect to both possible acquisition orientations. The new method is independent of Cas type, leader existence and location, and transcription orientation. CRISPR-evOr is thus particularly useful for arrays that other CRISPR orientation tools cannot predict confidently and to verify or resolve conflicting predictions from existing tools. CRISPR-evOr currently confidently predicts the orientation of 28.3% of the arrays in the considered subset of CRISPRCasdb, which other tools like CRISPRDirection and CRISPRstrand cannot reliably orient. As our tool leverages evolutionary information we expect this percentage to grow in the future when more closely related arrays will be available. Additionally, CRISPR-evOr provides confident decisions for rare subtypes of CRISPR arrays, where knowledge about repeats and leaders and their orientation is limited.

Long non-coding RNAs (lncRNAs) are emerging as key regulatory players of coding gene expression in eukaryotes. Here, we investigate the roles of the lncRNAs SVALKA (SVK) and SVALNA (SVN) in regulating CBF1 and CBF3 gene expression in Arabidopsis under cold stress conditions. We used Native Elongation Transcript Sequencing, CRISPR-Cas9 deletion strategies, and RT-qPCR to analyze the transcriptional dynamics and regulatory mechanisms of SVK and SVN. Our results demonstrate that SVK functions as a cis- and trans-acting lncRNA, regulating both CBF1 and CBF3 through RNAPII collision and chromatin remodeling, while SVN serves a cis role by negatively regulating CBF3 via a RNAPII collision mechanism. We identified isoforms of SVK, originating from distinct transcription start sites and undergo alternative splicing to adapt structural stability, crucial for their regulatory functions. This study elucidates the complex interplay of lncRNAs in gene regulation, highlighting their essential roles in modulating responses to environmental stresses. Our findings contribute to a deeper understanding of the mechanisms underlying lncRNA functionality and their significance in gene regulatory networks in eukaryotes.

Freezing behavior, characterised by attentive immobility as a reaction to a perceived threat, is widely studied in the context of fear, anxiety and stress. To uncover the genetic factors underlying this behavior, we conducted a genome-wide association study (GWAS) in kennel-housed beagle dogs. Our analysis identified a single-nucleotide polymorphism (SNP) in intron 5 of the KCNQ3 gene on chromosome 13 associated with freezing behavior in response to unfamiliar environments and people. To validate this finding, we used a zebrafish model, where CRISPR/Cas9-induced kcnq3 deficiencies led to heightened fear and arousal in two behavioral tests. KCNQ3 is implicated in several neurodevelopmental and psychiatric disorders, and our results highlight its evolutionarily conserved role in modulating fear responses. In dogs, an enriched environment can mitigate the adverse effects of KCNQ3 deficiency by reducing threat perception, highlighting the role of gene-environment interactions in shaping behavioral responses.

Abstract Endometrial carcinoma (EC), the most common gynecologic cancer type, encompasses multiple molecular subtypes that have consistent prognostic values and are being adopted in clinical practice to guide treatment decisions. However, it remains unclear whether each of these molecular subtypes have unique therapeutic vulnerabilities that can be exploited for advancing the management of ECs. Through analyzing the genomic features of a panel of 39 EC cell lines, we identified multiple tumor cell lines representing each molecular subtype. Histologic and immunochemical analyses of xenografted tumors from these cell lines confirmed their resemblance of cognate primary EC molecular subtypes, both by histology and the protein expression status of mismatch repair genes, p53 and SWI/SNF members in corresponding subtypes. Further investigation of the publicly available genome-wide CRISPR data for EC cell lines identified multiple specific genetic vulnerabilities in mismatch repair-deficient, p53-abnormal and ARID1A and ARID1B-dual deficient EC cell lines, respectively. Particularly, ARID1A and ARID1B-dual deficient EC cells selectively rely on mitochondria oxidative phosphorylation in vitro and in vivo . Therefore, our study demonstrates the utility of EC cell line models for uncovering and validating therapeutic vulnerabilities of each EC molecular subtype.

Recurrent breast cancer accounts for most disease-associated mortality and can develop decades after primary tumor therapy. Recurrences arise from residual tumor cells (RTCs) that can evade therapy in a dormant state, however the mechanisms are poorly understood. CRISPR-Cas9 screening identified the transcription factors SOX5/6 as functional regulators of tumor recurrence. Loss of SOX5 accelerated recurrence and promoted escape from dormancy. Remarkably, SOX5 drove dormant RTCs to adopt a cartilage-dependent bone development program, termed endochondral ossification, that was confirmed by [18F]NaF-PET imaging and reversed in recurrent tumors escaping dormancy. In patients, osteochondrogenic gene expression in primary breast cancers or residual disease post-neoadjuvant therapy predicted improved recurrence-free survival. These findings suggest that SOX5-dependent mesodermal transdifferentiation constitutes an adaptive mechanism that prevents recurrence by reinforcing tumor cell dormancy.

The success of cancer immunotherapies is currently limited to a subset of patients, which underscores the urgent need to identify the processes by which tumours evade immunity. Through screening a kinome-wide CRISPR/Cas9 sgRNA library we identified MAP3K7 (TAK1) as a suppressor of CD8+ T cell mediated killing. We demonstrate that TAK1 acts as a cancer-intrinsic checkpoint by integrating signals from T cell-secreted TNF and IFNy effector cytokines to elicit a cytoprotective response. This cytoprotective response profoundly limits the anti-cancer activity these key effector molecules and completely abrogates bystander killing by perforin deficient T cells. Inhibition of the TAK1 checkpoint effectively redirects the combined TNF/IFNy pathway activation to promote inflammatory cell death via RIPK1 and Caspase-8 and simultaneously amplifies the output of the IFNy pathway, thereby priming cells for cytokine-induced cell death. Mechanistically, TAK1 deficiency led to proteasomal degradation of cFLIP, enhancing the formation of Complex II and subversion of other cytoprotective responses. Targeting the TAK1 checkpoint led to profound attenuation of tumour growth in immune competent mice, with minimal impact in immune deficient counterparts. Adoptive cell therapy led to preferential elimination of TAK1 deficient clones. Collectively, our study uncovers a cancer-intrinsic checkpoint controlled by TAK1 activity that switches TNF and IFNy responses from cytoprotective to apoptosis. Cancer cells exploit this to limit cell death in the presence of the cytotoxic lymphoctye cytokines TNF and IFNγ and therapeutic intervention can fully unleash the impact of these effector molecules both on the direct target and bystander cells. These findings highlight the clinical development of TAK1 biologics as a potential strategy to improve cancer immunotherapies through harnessing and enhancing the cytotoxic potential of CTL-derived cytokines.

Metastatic breast cancer (MBC) is a life-threatening disease with limited therapeutic options. The immune suppressive tumor microenvironment (TME) limits the potency of the antitumor immune response and facilitates disease progression and metastasis. Our current study demonstrates that p38α is a druggable target in the TME that regulates the outcome of the immune-tumor interaction. The study revealed that systemic blockade of p38α reduces metastasis, and this anti-metastatic response is negated by depletion of CD8+ T cells. Single-cell transcriptomic analysis of the immune-TME showed that pharmacological p38 inhibition (p38i) or tumor-specific inactivation of p38α by CRISPR/Cas9 (p38KO) resulted in a less exhausted and more activated CD8+ T cell phenotype. Immunophenotyping analyses demonstrated that p38 blockade reduced the expression of multiple inhibitory receptors on CD8+ T cells (i.e., PD-1, LAG-3, CTLA-4), indicating a reversal of immune exhaustion and enhanced immune activation systemically and in the TME. In contrast, p38 blockade did not exhibit inhibitory effects on T cells in proliferation assays in vitro and did not affect the proportion of regulatory T cells in vivo. The major negative impact of p38 blockade in vivo was on the myeloid populations, such as myeloid-derived suppressor cells (MDSCs) and tumor-associated macrophages (TAMs). Further, tumor p38α activity was required for the expression of cytokines/chemokines and tumor-derived exosomes with high chemotactic capacity for myeloid cells. Altogether, this study highlights a previously unrecognized p38α-driven pathway that promotes an immune suppressive TME and metastasis, and that therapeutic blockade of p38α has important implications for improving antitumor immunity and patient outcomes.

A recent study has shown that SKP2 inactivation can prevent cancer initiation by extension of total cell cycle duration without perturbing normal division, which suggests a new strategy for cancer prevention. However, direct in vivo evidence for human SKP2 on cancer initiation and prostatic microenvironment is still lacking and a prostate-specific SKP2 humanized mouse model is critical for developing prostate cancer immunoprevention approaches through targeting human SKP2. We therefore have established a prostate-specific human SKP2 (hSKP2) knock-in mouse model by a CRISPR knock-in approach. Overexpression of hSKP2, which is driven by an endogenous mouse probasin promoter, induces prostatic lesions including hyperplasia, mouse prostate intraepithelial neoplasia (mPIN), and low-grade carcinoma and increases prostate weights. Transcriptional profiling by RNA-sequencing analysis revealed significant gene expression alterations in epithelial to mesenchymal transition (EMT), extracellular matrix, and interferon signaling in the prostate of hSKP2 knock-in mice compared to wild-type mice. Single cell deconvolution showed an increase of fibroblasts population and a decrease of CD8+ T cell and B cell populations in the prostate of hSKP2-knock-in mice. Consistently with these results from the SKP2 humanized mouse, overexpression of hSKP2 in human prostate cancer PC3 cells markedly increased cell migration and invasion and induced the gene expression of EMT and interferon pathways, including FMOD, THY1, PFKP, USP18, IL15, etc. In addition, paired prostate organoids were derived from SKP2 humanized and wild-type mice for drug screening and validated by known SKP2 inhibitors, Flavokawain A and C1. Both of which selectively decrease the viability and alter the morphologies of organoids of hSKP2 knock-in rather than wild-type mice. Our studies provide a well-characterized prostate-specific hSKP2 knock-in mouse model and offer new mechanistic insights for understanding the oncogenic role of SKP2 in shaping the prostatic microenvironment during early carcinogenesis.

CRISPR (Clustered Regularly Interspaced Short Palindromic Repeats) systems are a fundamental defense mechanism in prokaryotes, where short sequences called spacers are stored in the host genome to recognize and target exogenous genetic elements. Viromics, the study of viral communities in environmental samples, relies heavily on identifying these spacer-target interactions to understand host-virus relationships. However, the choice of sequence search tool to identify putative spacer targets is often overlooked, leading to an unknown impact of downstream inferences in virus-host analysis. Here, we utilize simulated and real datasets to compare popular sequence alignment and search tools, revealing critical differences in their ability to detect multiple matches and handle varying degrees of sequence identity between spacers and potential targets. Finally, we provide general guidelines that may inform future research regarding matching, which is a common practice in studying the complex nature of host-MGE interactions.

Prenylnaringenin (PN) compounds, namely 8-prenylnaringenin (8-PN), 3′-prenylnaringenin (3′-PN), and 6-prenylnaringenin (6-PN), are reported to have several interesting bioactivities. This study aimed to validate a biosynthetic pathway for de novo production of PN in Escherichia coli. A previously optimized E. coli chassis capable of efficiently de novo producing naringenin was used to evaluate eleven prenyltransferases (PTs) for the production of PN compounds. As PT reaction requires dimethylallyl pyrophosphate (DMAPP) as extended substrate that has limited availability inside the cells, clustered regularly interspaced short palindromic repeats (CRISPR) and CRISPR-associated protein 12a (Cas12a) (CRISPR-Cas12a) was used to construct ten boosted DMAPP-E. coli strains. All the PTs, in combination with the naringenin biosynthetic pathway, were tested in these strains. Experiments in 96-well deep well plates identified twelve strains capable of producing PN. E. coli M-PAR-121 with the integration of the 1-deoxy-D-xylulose-5-phosphate synthase (DXS) gene from E. coli (EcDXS) into the lacZ locus of the genome (E. coli M-PAR-121:EcDXS) expressing the soluble aromatic PT from Streptomyces roseochromogenes (CloQ) and the naringenin biosynthetic pathway was selected as the best producer strain. After optimizing the production media in shake flasks, 160.57 μM of 3′-PN, 4.4 μM of 6-PN, and 2.66 μM of 8-PN were obtained. The production was then evaluated at the bioreactor scale and 397.57 μM of 3′-PN (135.33 mg/L) and 25.61 μM of 6-PN (8.72 mg/L) were obtained. To the best of our knowledge, this work represents the first report of de novo production of PN compounds using E. coli as a chassis.

Cas9 provides a powerful tool to interrogate DNA repair and to introduce targeted genetic modifications. However, a major challenge of Cas9-based editing in human embryos is the occurrence of chromosomal abnormalities caused by Cas9 cleavage. Furthermore, mosaicism - different genetic outcomes in different cells, prevent accurate genotyping using a single embryo biopsy. Through timed analysis of editing outcomes during the first cell cycle and timed inhibition of Cas9 using AcrIIA4, we show that most edits occur at least 12 hours post Cas9 injection and therefore after the first S-phase. This timing limits the ability to achieve uniform editing across cells. We found that segmental chromosomal abnormalities and the consequential loss of heterozygosity are common at Cas9 cleavage sites throughout the genome, including at MYBPC3 and at CCR5 loci, for which this has not previously been reported. Surprisingly, inhibiting Cas9 activity 8-12 hours before mitosis does not eliminate chromosomal aneuploidies. This suggests that double-strand break (DSB) repair in human embryos is exceedingly slow, with breaks remaining unrepaired for many hours. Thus, the timing of DSB induction and repair relative to the first S-phase and the first mitosis is intrinsically limiting to preventing mosaicism and maintaining genome stability in embryonic gene editing.

RNA-guided CRISPR nuclease Cas9 cannot reliably differentiate between single nucleotide variations (SNVs) of targeted DNA sequences determined by their guide RNA (gRNA): they typically exhibit similar nuclease activities at any of those variations, unless the variation occurs with specific sequence contexts known as protospacer adjacent motifs (PAMs). Our approach, "TOP-SECRETS," generates gRNA variants that allow Cas9 ribonucleoproteins (RNPs) to reliably discriminate between healthy and disease-associated SNVs outside of PAMs.

CRISPR-Cas9 guide-RNA design tools can be used to identify guide-RNAs that target single human genomic loci. However, these approaches limit their effect to a single locus. Here, we generate a database of potential individual guide-RNAs that target multiple sites in the genome at once. All guide-RNAs in this database are curated with on- and off-target quantification and enrichment scores for genomic elements such as gene elements, regulatory elements, repetitive elements, and transcription factor motifs. This tool enables rationale mass targeting of genomic loci with a single guide for functional studies. We created a web-app for user-friendly guide-RNA selection (https://modreklab.shinyapps.io/guiderna/).

Cancer therapy has evolved dramatically over the past few decades, progressing from traditional treatments such as surgery, chemotherapy, and radiation therapy to more advanced approaches that target the cellular and molecular mechanisms underlying cancer. Understanding these mechanisms is crucial for developing more effective. While surgery, chemotherapy, and radiation therapy remain the cornerstones of cancer treatment, they are often associated with significant side effects and limited specificity. These treatments work by targeting rapidly dividing cells, but they cannot distinguish between cancerous and normal cells, leading to collateral damage. Cancer is fundamentally a disease of cellular and genetic dysregulation. Understanding the cellular and molecular mechanisms that drive cancer progression is essential for developing targeted therapies that can more precisely attack cancer cells while sparing normal cells. Signal transduction pathways regulate various cellular processes, including growth, differentiation, and survival. In cancer, these pathways are often dysregulated, leading to aberrant cell behavior. For example, the PI3K/AKT/mTOR pathway is frequently activated in cancer, promoting cell growth and survival. Combining different types of therapies can enhance their effectiveness and overcome resistance. For example, combining targeted therapies with immunotherapy or traditional treatments can lead to better outcomes. Researchers are continually exploring new combinations to find the most effective strategies. The field of cancer therapy is rapidly evolving, with ongoing research into new molecular targets, biomarkers for early detection, and strategies to overcome resistance. Advances in technologies such as CRISPR gene editing, artificial intelligence, and personalized medicine are poised to revolutionize cancer treatment. In conclusion, understanding the cellular and molecular mechanisms of cancer is crucial for developing more effective and less toxic therapies. While traditional treatments have their limitations, targeted therapies and new approaches offer hope for better outcomes and improved quality of life for cancer patients. Continued research and innovation are essential to conquer this complex and formidable disease.

Bacteria can encode dozens of different immune systems that protect cells from infection by mobile genetic elements (MGEs). Interestingly, MGEs may also carry immune systems, such as CRISPR-Cas, to target competitor MGEs, but it is unclear when this is favoured by natural selection. Here, we develop and test novel theory to analyse the outcome of competition between plasmids when one carries a CRISPR-Cas system that targets the other plasmid. Our model and experiments reveal that plasmid-borne CRISPR-Cas is beneficial to the plasmid carrying it when the plasmid remains in its host. However, CRISPR-Cas is selected against when the plasmid carrying it transfers horizontally, if the competitor plasmid encodes a toxin-antitoxin (TA) system that elicits post-segregational killing. Consistent with a TA barrier to plasmid-borne CRISPR-Cas, a bioinformatic analysis reveals that naturally occurring CRISPR-Cas-bearing plasmids avoid targeting other plasmids with TA systems. Our work shows how the benefit of plasmid-borne CRISPR-Cas is severely reduced against TA-encoding competitor plasmids, but only when plasmid-borne CRISPR-Cas is horizontally transferred. These findings have key implications for the distribution of prokaryotic defenses and our understanding of their role in competition between MGEs, and the utility of CRISPR-Cas as a tool to remove plasmids from pathogenic bacteria.

Abstract Probiotics ( lactic acid bacteria ) are widely used as microbial feed additives in livestock production and play an important role in preventing and treating animal diarrhea as well as regulating host immune function. In this study, lactic acid bacteria were isolated from the fresh feces of healthy adult female sheep, and their biological characteristics were analyzed. Based on phylogenetic analysis, strain SSF2 was identified as Pediococcus pentosaceus . SSF2 exhibited tolerance to acid, bile salt concentrations, and simulated artificial gastrointestinal environments. The hemolysis test for SSF2 was negative, it was sensitive to commonly used antibiotics, and it demonstrated significant antibacterial and antioxidant activities, indicating its excellent probiotic potential. Whole-genome sequencing (WGS) was performed using the HiSeq 2500 platform and the PacBio system to explore the genetic characteristics of SSF2. The genome was revealed to consist of a circular chromosome and two plasmids, with sizes of 1,785,410 bp, 10,618 bp, and 57,766 bp, and GC contents of 37.23%, 34.95%, and 40.98%, respectively. The genome was predicted to contain five genomic islands, six prophages, and a potential CRISPR gene editing sequence. Functional annotation through databases such as COG, GO, and KEGG revealed that most genes are related to carbon metabolism, protein and amino acid metabolism, nucleotide metabolism, and membrane transport processes. This study indicates that an in-depth understanding of the functionality and genetic characteristics of Pediococcus pentosaceus SSF2 may enable the potential application of this strain in sheep feed supplements.

Development of new and improved tuberculosis (TB) chemotherapies is hampered by antibiotic resistance and drug tolerance by Mycobacterium tuberculosis (Mtb). Phenotypic drug tolerance, a phenomenon where Mtb populations can temporarily survive therapeutic antibiotic concentrations, represents a significant hurdle to TB treatment and is indeed one of the factors responsible for prolonged TB therapy. Assays that can identify compounds with improved efficacy against drug tolerant Mtb are urgently required to improve TB treatment regimens. Here, we report the development of a 96-well plate assay capable of identifying anti-Mtb drugs with activity against drug tolerant Mtb in physiologically relevant intracellular environments within macrophages. Primary murine macrophages modified either by immunological activation or specific CRISPR/Cas9 gene knockouts to generate tolerance-inducing environments were infected with an Mtb strain constitutively expressing luciferase. Following drug exposure, differences in bacterial survival were measured by bacterial outgrowth after lysis of the host macrophages. By monitoring Mtb luciferase in infected macrophages before, during and after drug treatment, we confirmed earlier observations that host immune stresses trigger induction of drug tolerance. However, while host stresses induced tolerance against some anti-TB compounds, the same host stresses were synergistic with other anti-TB drugs. Our assay provides the ability to profile the activities of anti-TB drugs on bacteria in intracellular host environments which is critical to the rational design of drug combinations that provide optimal coverage of the Mtb sub-populations in the infected host.

The protein tyrosine phosphatases (PTPs) TCPTP, PTPN22, and SHP1 are critical regulators of the activating phosphotyrosine (pY) site on the initiating T cell kinase, LckY394. Still, the broader implications of these phosphatases in T cell receptor (TCR) signalling and T cell biology remain unclear. By combining CRISPR/Cas9 gene editing and mass spectrometry, we evaluate the protein- and pY-level effects of TCPTP, PTPN22, and SHP1 in the Jurkat T cell model system. We find that deletion of each phosphatase corresponds to unique changes in the proteome of T cells, with few large-scale changes to TCR signalling proteins. Notably, PTPN22 and SHP1 deletions have opposing effects on pY abundance globally, while TCPTP deletion modestly elevates pY levels. Finally, we show that TCPTP is indirectly involved in Erk1/2 positive feedback to the TCR. Overall, our work provides evidence for alternative functions of three T cell phosphatases long thought to be redundant.

Melanocytes reside in diverse microenvironments that influence their susceptibility to oncogenic transformation, however, studying rare melanoma subsets has been hindered by the lack of suitable animal models. We developed a primary, immune-competent zebrafish model to study uveal melanoma (UM), utilizing choroidal-targeted injection and electroporation of plasmids containing human GNAQ Q209L and CRISPR/Cas9 cassettes for tumor suppressor gene deletion. Single-cell transcriptional profiling of genetically identical eye- and skin-derived tumors revealed distinct oncogenic pathways, highlighting the importance of studying melanoma subtypes in their correct anatomical context. Additionally, we identified a population of tfec - and pax3a -expressing melanocyte progenitor cells in mitfa -deficient embryos and adult zebrafish eyes, which were highly susceptible to GNAQ-driven transformation. While previous studies have linked mitfa deficiency to accelerated UM onset, our findings suggest that an expanded progenitor population in mitfa -deficient animals drives this susceptibility. Our study establishes a critical role for Mitfa-independent melanocyte progenitors in UM pathogenesis.

Background: /Objectives: Notable similarities in lipid metabolism exist between human and golden Syrian hamster relative to most other rodents. A model for β-thalassemia in hamster was sought via knocking out the hemoglobin β-chain (HBB) gene. There are two HBB genes, as well as seven β-like alleles, predicted in the hamster genome, yet none have been functionally characterized. Methods: To develop a β-thalassemia hamster model and genetically interrogate the functions of the HBB genes in the hamster, we employed CRISPR/Cas9-mediated gene targeting technique and successfully knocked out one of the two hamster HBB genes. Results: Surprisingly, mass spectrometry analysis of the hemolysates from wild type, heterozygous, and homozygous knockout (KO) hamsters showed no changes in the hemoglobin β-chains at protein level. This indicates that the HBB gene that we chose to target does not code for proteins. Interestingly, lipid oxidation during storage was elevated in leg muscle of homozygous KO female hamsters compared to wild type females (P&lt;0.05). Conclusions: Our study provided a path toward developing a hamster β-thalassemia animal model, and related findings suggest an effect of a non-translated HBB gene on oxidative stress. In addition, mass spectrometry provides a way to quickly identify non-protein-coding-genes in species where genomic/transcriptomic annotation is not fully developed.

CRISPR-Cas adaptive immunity systems provide defense against mobile genetic elements and are often countered by diverse anti-CRISPR (Acr) proteins. The Type IE CRISPR-Cas of Escherichia coli K12 has been a model for structural and functional studies and is a part of the species’ core genome. However, this system is transcriptionally silent, which has fueled questions about its true biological function. To clarify the role of this system in defense, we carried out a census of Acr proteins found in Enterobacterales and identified AcrIE9 as a potent inhibitor of the E. coli K12 Type IE CRISPR-Cas system. While sharing little sequence identity, AcrIE9 proteins from Pseudomonas and Escherichia both interact with the Cas7 subunit of the Cascade complex, thus preventing its binding to DNA. We further show that AcrIE9 is genetically linked to AcrIE10, forming the most widespread anti-CRISPR cluster in Enterobacterales , and this module often co-occurs with a novel HTH-like protein with unusual architecture.

Rice (Oryza sativa L.) is a vital global crop with a predominant presence in Asia, including Thailand. However, it faces a significant threat from bacterial blight disease (BB), primarily caused by Xanthomonas oryzae pv. oryzae (Xoo). This research aims to provide an insights into the genetic virulence and variation of Xoo strains isolated in Thailand. Our phylogenetic analysis unveils that the 20 Thai strains align with the Asian strains, setting them apart from African and USA strains. Remarkably, the Average Nucleotide Identity (ANI) values, in comparison to the Xanthomonas oryzae type strain 35933 (XO35933), consistently exceed 99%. These strains can be classified into three assigned ribosomal sequence types (rST). Our investigation into the pangenome and the phylogenetic relationships of these 20 Xoo genomes reveals a diverse genetic landscape, with the pangenome comprising 11,872 orthologous gene clusters, of which roughly 30% form the core genome. Notably, all of these genomes exhibit the presence of a CRISPR-Cas I-C array, indicative of their adaptive immune mechanisms. All strains belonged to BXO1 type LPS cassette with high identity. Furthermore, our analysis identifies two distinct types of plasmids, namely, Xanthomonas oryzae pv. oryzicola strain GX01 plasmid pXOCgx01 (A46, A57, A83, A112, D, and E) and the Xanthomonas oryzae strain AH28 plasmid pAH28 (A97). This genomic resource will be valuable for advancing research on surveillance, prevention, management, and comparative studies of this critical pathogen in the future.